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Allosteric trans-splicing group i ribozyme whose activity of target-specific RNA replacement is controlled by theophylline

a technology of allosteric transsplicing and ribozyme, which is applied in the field of allosteric transsplicing group i ribozyme whose target, can solve the problems of not maximizing the desired effect of treatment diseases, many problems to be solved in the existing gene therapy technology, and the inability to maximize the therapeutic effect of diseases,

Inactive Publication Date: 2011-01-06
IND ACADEMIC COOP FOUND DANKOOK UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]These allosteric trans-splicing ribozymes may be used to develop a system that is able to target certain disease-specific RNA and artificially control the replacement into therapeutic gene RNA by using exogenous factors such as small molecules to activate the functions of the ribozyme. Also, a novel concept of specific and reversible gene therapy technologies may be developed by artificially controlling the expression of therapeutic genes in an infected cell-specific manner (FIG. 1).

Problems solved by technology

However, there are many problems to be solved in the existing gene therapy technologies.
Therefore, the virus particles do not include a variety of genetic elements that may control transporter genes in and of themselves, and do not maximize the desired effects for the treatment diseases.
Also, the promoters used for gene expression and the like may undesirably activate other kinds of promoters, and also may increase the expression of different genes (for example, protooncogenes) of cells, to which the genes are transferred, by changing the chromatin structure.
When the mutant gene products do have a dominant negative effect, their therapeutic effects may not be maximized by the conventional methods.

Method used

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  • Allosteric trans-splicing group i ribozyme whose activity of target-specific RNA replacement is controlled by theophylline
  • Allosteric trans-splicing group i ribozyme whose activity of target-specific RNA replacement is controlled by theophylline
  • Allosteric trans-splicing group i ribozyme whose activity of target-specific RNA replacement is controlled by theophylline

Examples

Experimental program
Comparison scheme
Effect test

reference example 1

Preparation of Substrate (hTERT) RNA

[0070]To prepare target RNA, a pCl-neo vector (exon 1-2) containing a −1st to +218th DNA sequence of the hTERT was PCR-amplified with a primer (5′-GGGGAATTCTAATACGACTCACTATAGGGCAGGCAGCGCTGCGTCCT-3′) set forth in SEQ ID NO: 9 and a primer (5′-CGGGATCCCTGGCGGAAGGAGGGGGCGGCGGG-3′) set forth in SEQ ID NO: 10, thus to prepare a DNA fragment encoding hTERT RNA. The DNA fragment thus prepared was transcribed in vitro into RNA. A DNA template (3 μg), a 10× transcription buffer, 10 mM DTT (Sigma), 0.5 mM ATP, GTP, CTP and UTP (Roche), an 80U RNase inhibitor (Kosco), a 200U T7 RNA polymerase (Ambion) were added, and DEPC-H2O was added to a final volume of 100 μl, and then mixed. Then, the resulting mixture was reacted at 37° C. for 3 hours, and further treated with 5U DNase I (Promega) at 37° C. for 30 minutes to completely remove the DNA template. RNA was purified through the phenol extraction (pH 7.0) and ethanol precipitation, and separated on 6% denatur...

reference example 2

Cloning of Theophylline-Dependent hTERT Targeting Trans-Splicing (T / S) Aptazyme

[0071]As a basic trans-splicing ribozyme backbone used to develop an allosteric ribozyme, group I intron ribozyme, which specifically recognizes a +21 nt site of hTERT and has P1, P10 and extended IGS to which 300 nt anti-sense sequence against target RNA is annealed, was used (Kwon, B. S., Jung, H. S., Song, M. S., Cho, K. S., Kim, S. C., Kimm, K., Jeong, J. S., Kim, I. H., and Lee, S. W. 2005, Specific regression of human cancer cells by ribozyme-mediated targeted replacement of tumor-specific transcript. Mol. Ther. 12: 824-834; Hong, S. H., Jeong, J. S., Lee, Y. J., Jung, H. I., Cho, K. S., Kim, C. M., Kwon, B. S., Sullenger, B. A., Lee, S. W.*, and Kim, I. H.* 2008, In vivo reprogramming of hTERT by trans-splicing ribozyme to target tumor cells. Mol. Ther. 16: 74-80).

[0072]A theophylline aptamer was cloned into either or both of P6 and P8 domains of the hTERT targeting ribozyme by means of a communica...

reference example 3

Preparation of Theophylline-Dependent hTERT Targeting T / S Aptazyme RNA

[0074]The DNA sequence of the theophylline-dependent hTERT targeting T / S aptazyme prepared in Reference example 2 was PCR-amplified with a primer set forth in SEQ ID NO: 16 (5′-GGGGAATTCTAATACGACTCACTATAGGCAGGAAAAGTTATCAGGCA-3′) including a T7 polymerase promoter and a primer set forth in SEQ ID NO: 17(5′-CCCAAGCTTGCGCAACTGCAACTCCGATAA-3′) that is annealed with the midway site of the 3′ exon of the ribozyme. In this case, an increased amount of a DNA template (3 μg) and NTP (1.5 mM) was used to prevent a self-splicing reaction as much as possible. Then, a 1× splicing buffer (40 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 10 mM DTT and 4 mM spermidine), 0.5 mM ATP, GTP, CTP, UTP (Roche), an 80U RNase inhibitor (Kosco), a 200U T7 RNA polymerase(Ambion) were added, and DEPC-H2O was added to a final volume of 100 μl and then mixed. Then, the resulting mixture was transcribed at 37° C. for 3 hours, and further treated with 5U DNa...

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Abstract

Provided is an allosteric trans-splicing group I ribozyme whose target-specific RNA replacement activity is controlled by theophylline, wherein the hTERT-targeting trans-splicing ribozyme recognizes mRNA of human telomerase reverse transcriptase (hTERT) as a cancer-specific RNA transcript to bind a theophylline aptamer to an hTERT target trans-splicing ribozyme via a communication module, the hTERT target trans-splicing ribozyme having a verified trans-splicing ability. The allosteric trans-splicing group I ribozyme may be useful to selectively diagnose only cancer cells that express target hTERT RNA, or induce their apoptosis since the activity of the allosteric trans-splicing group I ribozyme is dependently controlled by theophylline to correct target hTERT RNA by the trans-splicing reaction.

Description

CROSS REFERENCE TO RELATED CASES[0001]This application is a 371 National Stage of PCT / KR2008 / 007440, filed Dec. 16, 2008, which claims priority from Korean Patent Application No. 10-2008-0028483, filed Mar. 27, 2008, the entire disclosures of which are incorporated herein by reference in their entirety.TECHNICAL FIELD[0002]The present invention relates to an allosteric trans-splicing group I ribozyme whose target-specific RNA replacement activity is controlled by theophylline.BACKGROUND ART OF THE INVENTION[0003]There have been ardent attempts to develop a gene therapy technology as a new therapy technology to treat incurable human diseases by studying the molecular genetic causes and factors of the incurable human diseases caused by the gene mutations. However, there are many problems to be solved in the existing gene therapy technologies.[0004]Gene therapy that has been widely used to treat genetic diseases is devised by transferring a normal gene corresponding to a mutant gene to...

Claims

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Application Information

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IPC IPC(8): A61K48/00C12Q1/68C12N15/63C07H21/02
CPCC12N15/111C12N15/1137C12N2310/124C12N2310/1241G01N2333/9005C12N2310/3519C12Y207/07049G01N33/573C12N2310/16A61P35/00A61P43/00C12Q1/6886
Inventor LEE, SEONG WOOKJANG, SUN YOUNGKIM, JU HYUN
Owner IND ACADEMIC COOP FOUND DANKOOK UNIV
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